Separation Of Flavonoids In Natural Products Using High-speed Countercurrent Chromatography: Separation Of Flavonoids From Soybean, Ampelopsis Grossedentata And Salix Alba

Flavonoids possess several bioactivities such as anticancer, preventing angiocardiopathy and antibiosis. The present paper mainly aims to the preparative separation of the flavoniods in extract of soybean, leaves extract of Ampelopsis grossedentata and bark extract of Salix alba.Four isoflavone components were purified from soybean extract by high-speed counter-current chromatography (HSCCC). Two types of multilayer coil separation columns were used: a small column made of standard 2.6-mm I.D. PTFE (polytetrafluoroethylene) tubing with a 260-ml capacity and a large column of convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Separation was performed with a two-phase solvent system composed of hexane-ethyl acetate-1-butanol-methanol-acetic acid-water (1:2:1:5:1, v/v) by eluting the lower aqueous phase at 2ml /min (small column) and 5ml /min (large column) at a revolution speed of 700rpm. From 500 mg of crude sample the small column yielded 33mg of daidzin, 41mg of genistin, 27mg of 60-0-malonyldaidzin and 24mg of 6'-0-malonylgenistin. The large convoluted column separated, from 3g of crude sample, 203mg of daidzin, 241mg of genistin, 158mg of 6'-Omalonyldaidzin and 135mg of 60-O-malonylgenistin all at over 90% purity.Purification of dihydromyricetin from an extract (16g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:2:4, v/v) 11.3g of dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9h. Separaton of myricetrinin was conducted through two steps. First, the extract of leaves was crystallized with water to remove most of dihydromyricetin in order to obtain the extract with relative myricetrinin content. Then, the extract was separated using HSCCC with water:methanol:ethyl acetate:n-hexane(4:3:6:l, v/v) as solvent system. At the givenOHOHOHO(1)(2)condition, 500mg extract was successfully separated to yield 63mg myricetrinin. The chemical structures of dihydromyricetin and myricetrinin were confirmed by means of ESI-MS-MS, and NMR analysis.The flavonoids in bark extract of Salix alba were separated at preparative scale using high-speed countercurrent chromatography (HSCCC). In each separation, 1.0 g crude extract was separated to yield pure 120mg eriodictyol (I), 29.5 mg 5,7-dihydroxychromone (II) and 50 mg naringenin (III), respectively, while water-methanol-ethyl acetate-n-hexane (3:2:2:2, v/v) was used for a two-phase solvent system of the HSCCC separation. The chemical structures of three flavonoidswere confirmed by means of ESI-MS-MS, and NMR analysis.OH4. OH...